The overarching goal of this project is to reveal the mechanisms that ensure error-free segregation of chromosomes during mitosis. While higher levels of segregation errors are lethal, relatively infrequent mis- segregation of individual chromosomes underlies 'chromosomal instability' (CIN), a hallmark of cancer transformation. Therefore, identifying the exact cause(s) of CIN and finding a way to attenuate the rate chromosome missegregation will likely lead to novel strategies for selective elimination of cancer cells. Toward this goal we are employing sophisticated imaging in conjunction with molecular and cell-biology techniques to characterize the 'mitotic spindle', a self-assembling molecular machine that enacts chromosome segregation. In the current funding period we plan to achieve three objectives: 1) Reconstruct 3-D architecture of the spindle in normal and CIN cells via a novel approach based on super-resolution light microscopy. Comparative analyses of spindle organization in normal vs. CIN cells will reveal the structural foundation of chromosome loss. Specifically, we will test the hypothesis that some chromosomes lack direct connections to spindle pole and that these chromosomes are more numerous in CIN cells. 2) Characterize the structural reorganization of kinetochores (macromolecular assemblies that attach chromosomes to spindle microtubules) during the transition from the initial 'lateral' to the final 'end-on' attachments. In spite of their importance, the ultrastructure of lateral attachments has not been described due to technological limitations. We will capitalize on a novel approach that involves direct correlation of multi-color light-microscopy with high-resolution electron tomography conducted on the same kinetochore. This approach will allow us test the hypothesis that the same molecular machinery (Hec1) mediates both lateral and end-on attachments and also reveal reorganization of the outer plate that underlies satisfaction of the mitotic checkpoint. 3) Test the hypothesis that mild inhibition of dynein, benign in normal cells, selectively suppresses mitosis in CIN cells. This idea stems from the preliminary data that a significant number of chromosomes in CIN cells lack a direct attachment to the spindle poles and these chromosomes rely on dynein-mediated transport for segregation. We will systematically characterize the effects of dynein inhibitors on cell proliferation, levels of cell death, patternsof chromosome movement, and mechanisms of spindle assembly in normal vs. CIN cells. If our hypothesis is proven correct, partial inhibition of dynein will emerge as a powerful therapeutic approach.